BLADDER CARCINOMA CELL LINES AND METHODS Human bladder cells and culturing

1. #4320: Growth Inhibitory Effects of ZD1839 (‘Iressa’) on Human Bladder Cancer Cell LinesAxel Meye*, Ulrike Fiedler, Kathrin Kunert, Andres Melchior, Susanne Füssel, Manfred P. WirthDepartment of Urology, Technical University Dresden, F. R. of Germany* email: axel.meye@mailbox.tu-dresden.de; ‘Iressa’ is a trade mark of the AstraZeneca group of companies. 1,4 HT-1376 • RESULTS II • As expected, cells stimulated with EGF had a lower proportion of cell population in G1 and G2/M phase in comparison to untreated cells (Table 1a). Additional treatment with ZD1839 abolished the EGF-induced alterations of cell cycle distribution in HT-1376 (Table 1b). • Simultaneously, ZD1839-treated HT-1376 and EJ28 cells showed an increased rate of apoptosis (9.9% and 14.7%, respectively) in comparison to the untreated control (Table 2) INTRODUCTION & OBJECTIVES Importance of EGFR in bladder cancer biology Bladder cancer cells frequently exhibit an increased number of functional EGFR in comparison to normal urothelium. EGFR plays an important role in bladder cancer motility (Theodorescu et al. 1998a and 1998b). In human bladder cancer upregulation of EGFR often is correlated with increasing malignancy (Neal et al. 1990, Messing 1990, Mellon et al. 1995, and 1996, Bue et al. 1998, Turkeri et al. 1998, Luikkonen et al. 1999). Antiproliferative Activity of the specific EGFR tyrosine kinase inhibitor ZD1839 One of the intensively studied tyrosine kinases is the plasma membran glycoprotein EGFR. The different tumor biological activites of the EGFR in proliferation, angiogenesis and metastasis together with the association of tumor patients (with an intratumoral EGFR overexpression) with poor prognosis implies the efficient strategy to inhibit EGFR in cancer therapy (Woodburn 1999). ZD1839 (‘Iressa’) is an orally active selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that shows reversible antitumor activity in a broad range of established carcinoma cell lines and tumour xenografts (Ciardiello et al. 2000a, Sirotnak et al. 2000). The present study evaluated the antiproliferative activity of ZD1839 on bladder cancer cells in vitro. Four cell lines (EJ28, 5637, J82, HT-1376) derived from human transitional cell carcinoma (TCC) with different EGFR expression levels were investigated. 1,2 1,0 0,8 OD 450nm 0,6 0,4 0,2 negative cells with only EGF 0.01 0.05 0.1 0.5 1 control 1,5% FCS ZD1839 concentration [µM] Fig. 1: Effect of different ZD1839 concentrations (0.1-1 µM) on proliferation of HT-1376 cells. [Standard deviations are indicated. As controls served only medium (without cells), cells with FCS (1.5%) and EGF-stimulated cells (5 ng/mL) after 3 days (blue columns), 6 days (red colums) and 9 days (yellow colums).] • RESULTS I • Initial treatment experiments with various ZD1839 concentrations (0.5-25 µM) and under normal cell culture conditions (10% FCS) showed a significant inhibition of proliferation for all for cell lines (Wst-1 assays, day 3 to day 9). Complete growth inhibition was found for ZD1839 doses >10 µM (not shown). Most efficient reduction of proliferation was determined for both cell types with a high EGFR protein level (HT-1376 and EJ28). To study the ZD1839 specific effects more in detail, further experiments were carried out especially with the latter both cell types, and with reduced FCS content in the medium and at lower inhibitor dosage. • The detailed results of these experiments under optimized conditions (minimal FCS and optimal EGF concentrations) are represented in the figures and tables. They can be summarized as following: • dose-dependent and specific inhibition of EGF-stimulated tumour cell proliferation for all for TCC lines (Fig. 1 & 2) under optimized culturing conditions (minimal FCS-c & optimal EGF-c) • cell-type specific IC50 for ZD1839 (HT-1376<EJ28<5637<J82) correlated to the EGFR protein level evaluated by Western blotanalysis (HT-1376>EJ28 >5637/J82; data not shown) (Melchior et al., manuscript in preparation) • under confluent culture conditions tumour cells with a low viability showed a higher resistance to the same ZD1839 concentration as determined for cells growing in the log phase (data not shown) • initial data on proliferation rates of HT-1376 cells in long-term culturing experiments (3-14 days after treatment) indicated that the inhibitory effect of ZD1839 was reversible at relatively low concentrations (0.025-0.1µM) (Fig 3). BLADDER CARCINOMA CELL LINES AND METHODS Human bladder cells and culturing The cell lines J82 (ATCC #HTB-1), HT-1376 (ATCC #CRL-1472) and 5637 (ATCC #HTB-9) originated from human bladder cancers obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA), the EJ28 cell line was a gift [Block et al. 1993]. The cells were propagated as recommended by the supplier and without addition of antibiotics. For EGFR stimulation only reduced percentage of FCS (1% for EJ28 cells, 1.5% for J82 and HT1376, and 2% for 5637) was added to the culture medium. First experiments were carried out to determine the optimal EGF concentration for each cell type (to mediate a efficient EGFR stimulation): 1 ng/mL EGF for EJ28, 5 ng/mL EGF for HT-1376 (data not shown). EGF, and EGFR inhibitor ZD1839 (‚IRESSA‘) treatment Purified recombinant human EGF was purchased from R&D Systems (Wiesbaden, Germany). Clinical grade ZD1839 (‚Iressa‘) was kindly provided by AstraZeneca Pharmaceutical GmbH (Plankstadt, Germany). WST-1 assay, apoptosis test, and cell cycle analysis in vitro To determine the proliferation and viability of cells a colorimetric WST-1 assay (Boehringer Mannheim, Germany) based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells was applied according to the manufacturs protocols. Briefly, cells were analysed 3, 6 and 9 days after seeding of defined cell numbers (for treatment experiments with ZD1839: EJ28 with 400 cells/well, 5637 with 1000 cells/well, J82 with 600 cells/well, amd HT1376 with 700 cells/well) in 96-well plates as indicated. Cells were incubated with 20 µl WST-1 reagent for two hours at 37 °C. The detection was carried out at 450 nm at a conventional microplate reader. To analyze the cell cycle approximately 5x 105 cells were washed in 70% ethanol and stored at 4 °C until measurement. The DNA content was measured through incorporation of propidium iodide using a standard protocol (CycleTEST PLUS DNA Reagent Kit; Cell Cycle Becton Dickinson, Heidelberg, Germany). DNA distribution of the cell populations after described treatment procedures was analyzed by flow cytometry in a FACScan (Becton Dickinson). The rate of apoptosis was determined by the ApoAlert Caspase-3 colorimetric assay (Clontech, Heidelberg, Germany). Briefly, cells were seeded into 6-well plates. After appropiate treatment, the cells were washed once with PBS, and lysis buffer was added. After an incubation step for 30 min, the supernatant was recovered and assayed as described by the manufactures. The data for evaluation of apoptosis and cell cycle analysis represent two independent experiments, respectively. Table 1 a) Influence of EGF stimulation experiments on cell cycle phase distribution for HT13-76 cells (standard deviations are indicated) b) Addition of ZD1839 to EGF-stimulated cells results in a reblockade of at the G1 arrest only in HT-1376 (with a high EGFR expression level), whereas the ZD1839 treatment for EJ28 has no remarkebly influence on the EGF-induced cell cycle alterations. 1,3 EJ28 2.d 1,2 3.d 1,1 4.d 6.d 1,0 0,9 0,8 OD 450 nm 0,7 0,6 Table 2 Difference in apoptosis rate in cell lines EJ28 and HT-1376 treated with EGF alone or with EGF and ZD1839 (specific concentrations of both are indicated). Both cell types showed a ZD1839-induced specific increase in apoptosis after 3 days in comparison to control (only EGF stimulation). Results of caspase assay (ApoAlert Caspase-3-colorimetric assay) were calculated as difference in caspase activity (PNA per hour [nmol]). 0,5 0,4 0,3 negative cells with only EGF 0.01 0.05 0.1 0.5 1 CONCLUSIONS Apoptosis measurements for TCC lines EJ28 and HT-1376 indicate a moderate apoptosis induction in the first three days after treatment with ZD1839 (Table 2). The treatment with ZD1839 abolished the EGF-induced alterations of cell cycle distribution only in HT-1376 (Table 1). Treatment with EGFR inhibitors could cause a cytotoxic effect generally with cell cycle arrest at the G1 checkpoint. However, there are also studies describing an induction of apoptosis in several tumor types after EGFR blockade (Tortora et al. 1999, Ciardiello et al. 2000a). The data presented clearly indicate the potential for EGFR/TK inhibition as a therapeutic approach to TCC where EGFR drive is important. Recent results of Cardiello et al. (2000a) indicate an additive, growth inhibitory effect of low doses ZD1839 in combination with various cytotoxic agents in vitro, including an increase of apoptotic cell death by approximately 2-3.5-fold. Moreover, Sirotnak et al. (2000) found that ZD1839 does not require high levels of intratumoral expression as determined for different tumor types (including prostate carcino-ma, lung carcinoma, vulvar carcinoma xenografts) and combinational treatment schedules with other cytotoxic chemotherapeutics. As described in the latter two studies we started in testing the antiproli-ferative activity of ZD1839 in combination with relevant cytotoxic agents for advanced bladder cancers (gemcitabine, cisplatin, taxol). 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